PROJECT SUMMARY Clostridium difficile is the most commonly reported nosocomial pathogen in the United States and an urgent public health threat worldwide. Over the past decade, incidence, severity, and costs associated with C. difficile infection (CDI) have increased dramatically; however, the factors that govern this broad spectrum of disease remain unclear. The primary risk factor for CDI is antibiotic use, which reduces colonization resistance to C. difficile by altering the resident gut microbiota. Interestingly, the major nosocomial pathogen vancomycin- resistant Enterococcus (VRE) shares numerous risk factors with C. difficile and these pathogens are commonly found together in the gastrointestinal tract. Moreover, colonization with VRE is associated with more severe C. difficile-associated disease and in subsets of patients, CDI increases the risk of bacteremia due to VRE. Despite these strong associations, little work has been done to explore the molecular interactions between Enteroccoci and C. difficile during infection and it is unclear what impact co-occurrence of these pathogens has on C. difficile-associated disease. Interspecies interactions can be attributed to the development of numerous polymicrobial infections, including CDI, and cross talk between bacteria is associated with exacerbation of several diseases. Enterococcus is associated with increased susceptibility to CDI and the gut microbiota of patients with CDI is highly enriched with Enterococci. Following excess Zn supplementation, commensal Enterococci are highly enriched in the microbiota, which leads to increased susceptibility to CDI, exacerbation of C. difficile-associated disease, and high levels of Enterococcus translocation to the liver. Preliminary data suggests that in culture, Enterococcus and C. difficile directly interact and this cross-talk alters virulence and behavior of these two pathogens. In this application, we propose to elucidate the interactions between Enterococcus and C. difficile and define the impact that these interactions have on susceptibility and severity of CDI. We hypothesize that (i) Enterococcus increases susceptibility to CDI by altering the landscape of the gut to enhance C. difficile colonization and (ii) co-occurrence of Enterococcus and C. difficile in the gastrointestinal tract facilitates cross-talk between these two important pathogens that increases persistence and exacerbates disease during CDI. We plan to test these three hypotheses through a series of integrated Specific Aims. First, we will dissect the relationship between Enterococcus abundance and susceptibility to C. difficile and determine the contribution of Enterococcus to C. difficile virulence in mice (Aim 1). We will then define how these pathogens interact during infection and examine the impact of bacterial cross-talk on disease (Aim 2). These experiments will elucidate the impact of VRE on susceptibility, severity, and recurrence of CDI and will substantially increase our understanding of how C. difficile interacts with other common pathogenic microbiota during infection. The insights gained by completion of this proposal will lay the foundation for novel therapeutics that aim to disrupt the molecular interactions that facilitate C. difficile infection.